Use of a mast cell activation or degranulation blocking agent in the manufacture of a medicament for the treatment of a patient subjected to thrombolyses

ABSTRACT

The invention concerns the use of a mast cell activation- or degranulation-blocking agent in the manufacture of a medicament for preventing and treating cerebral complications caused by thrombolytic treatment. The invention also relates to treatment of patients suffering from cerebral complications associated with thrombolysis. Further, the invention provides thrombolytic compositions comprising a mast cell degranulation-blocking and/or mast cell activation-blocking agent present in a therapeutically effective amount to prevent or reduce any cerebral complications caused by the active thrombolytic component.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 National Stage Entry of InternationalApplication No. PCT/FI2004/000072, filed Feb. 13, 2004, which claimspriority to U.S. Patent Provisional Application No. 60/446,990, filedFeb. 13, 2003.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to new therapeutic uses of knowncompounds. In particular, the invention relates to the use of mast cellactivating- and degranulation-blocking agents for preventing andtreating cerebral complications, such as reperfusion injury,inflammatory changes, brain edema, and similar damages occurring inconnection with thrombolytic treatment.

2. Description of Related Art

Mast cells are present in the brain and located typically inperivascular spaces. They contain substantial amounts of preformedpro-inflammatory, vasoactive, anticoagulant and proteolytic substances.These substances are contained within numerous intracytoplasmicgranules. They can synthesise a large number of additional substances.Generally, they protect parenchymal organs from exogenous, hazardousagents such as microbes and toxic or allergenic particles. The chymasefound in the granules can lyse vascular basal membrane constituents suchas fibronectin. Mast cells can influence the permeability of smallcerebral vessels, regulate blood circulation and prime immunologicalresponses. They can also mount an immediate host defence response byrapid degranulation, which can lead to hazardous anaphylactic and othersystemic bodily reactions. Mast cells have been found in mostparenchymal organs, including the brain and meninges as well asperitoneal organs such as the intestines.

Mast cells are targets of therapy to treat allergic reactions occurringin asthma or allergic conjunctivitis. Mast cells are also known to beactivated and degranulated during mechanical manipulations in the skinas well as trauma to the various parts of the body, and parenchymalorgans, but this has not been a basis for any therapeutic methods ormodifications of surgical procedures. There are currently no approvedmedical treatments, which would depend on specific modulation of themast cell function in the brain, but there have been speculations thatthey participate pathophysiologic events in migraine, multiplesclerosis, neuroendocrine reactions such as psychological stressreactions and Wernicke's encephalopathy, a rare brain disease caused bymalnutritional state. In the treatment and various clinicalinvestigations of cerebrovascular disease such as ischemic andhemorrhagic stroke, brain trauma, brain tumors or increased intracranialpressure, mast cells are not considered as participants of the diseasesor targets of therapeutic interventions.

Thrombolytic therapy has been the culprit of acute myocardial infarctionand massive pulmonary embolism for a decade. It is given often alreadyby the emergency dispatch medical personnel outside the hospital. It isalso used in acute occlusions of peripheral arteries to prevent limbnecrosis. Thrombolytic therapy with alteplase (recombinant tissueplasminogen activator, r-TPA) has recently been approved also for acuteischemic stroke in North America and Europe. Stroke thrombolysisnecessitates the rapid transport of patients into the hospital toreceive computed tomography to rule out a brain hemorrhage.

r-TPA is a serine protease leading to plasmin formation, which dissolvesfibrin fibres, a major constituent of arterial thrombi. Although theaction of r-TPA is considered relatively specific, and the specificityof thrombolytic substances is under constant research andpharmacological development, thrombolysis is associated with hemorrhagiccomplications such as gastrointestinal bleeding and hematomas of theparenchymal organs. Those occurring in the brain are among the mostdreaded and can be fatal. In acute myocardial infarctions, the rate ofintracranial bleeding is 0.5-1.0%, but in acute ischemic stroke thefrequency of parenchymal hematomas is 6-11%. An equal additional amountof patients undergo hemorrhagic transformation of the ischemic stroke,which, however, can occur also in patients who do not receivethrombolysis. When treating ischemic stroke patients with thrombolysis,the single most feared complication and factor to preclude treatment isthe possibility to cause serious intracranial hemorrhage with thetreatment. Furthermore, it can be associated with enhanced reperfusioninjury and brain edema. If these safety issues would be solved, a muchlarger fraction of stroke patients today would receive thrombolytictherapy.

Today, there are no substances in clinical use to reduce the rates ofhemorrhagic complications in thrombolysis. To prevent hemorrhages,arterial blood pressure is controlled and kept under 185/110 mmHg.However, perithrombolytic administration of antihypertensive compoundshas been associated with poor functional prognosis, which may relate toreduced cerebral perfusion. Patients who might have increased tendencyto bleed, such as those with reduced hemostasis, malignancies,anticoagulant therapy or recent biopsies or operations, are generallynot treated with thrombolytics to prevent hemorrhagic complications.

For the reasons, there is a great need for alternative or additionalmethods for preventing or treating brain edema and similar cerebraldamages caused by thrombolytic treatment.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a new use of mastcell degranulation-blocking and/or mast cell activation-blocking agents.

It is another object of the present invention to provide a novel methodfor treating and protecting patients subjected to thrombolytic treatmentfrom cerebral complications, such as brain edema, and for specificallyprotecting the brain-blood barrier.

It is a third object of the present invention to provide novelthrombolytic compositions.

These and other objects, together with the advantages thereof over theknown therapeutic uses, which shall become apparent from specification,which follows, are accomplished by the invention as hereinafterdescribed and claimed.

In connection with the present invention, it has been observed that mastcells are present in the brain tissue, surrounding small cerebralvessels in perivascular spaces, and increase in number, and commonlydegranulate, during and following induced cerebral ischemia. Theycolocalize with perivascular edema formation, early leukocyte emigrationfrom the blood circulation as well as erythrocyte extravasation. It istherefore believed that the initial response of stationary mast cells isin part causing later events leading to florid blood-brain barrierdamage and the entry of circulating inflammatory cells, which canaggravate the brain injury and disrupt the blood-brain barrier to largerparticles, such as erythrocytes.

The present inventors have discovered that preischemic administration ofmast cell stabilizer cromoglycate, which prevents mast celldegranulation, into the cerebral ventricles, prevented 39% of the brainedema observed already 3 hours after 60 minutes of middle cerebralartery occlusion in the rat. Furthermore, in connection with the presentinvention, the Inventors observed that a mast cell degranulating agent,compound 48/80, administered before reperfusion aggravated the brainedema by 89%. Furthermore, the Inventors observed that cerebralextravasation of plasma constituents was similarly influenced by thesame treatments in the ischemic brain area. Therefore, mast celldegranulation seems to participate in ischemic blood-brain barrierdamage, and could underlie in part the extravasation of blood cells andhemorrhagic transformation.

The inventors have also found that rat peritoneal mast cells ex vivo aredegranulated by alteplase solution. In the rat brains, degranulation ofcells consistent with mast cell morphology during a 90 minute ischemicperiod occurs in a subtotal manner up to 50%, and can be significantlyincreased both in the ischemic (up to 70%) and non-ischemic hemispheres(up to 60%) by systemically administered r-TPA infusion 5 minutes beforereperfusion. This associated with substantial hemorrhage formation inthe same rat brains. Furthermore, r-TPA infusion led to substantialneutrophil emigration into the brain parenchyma and adhesion within thecerebral microvessels, and the total number of neutrophils found in thetissue was up to 5-fold increased in the non-infarcted hemisphere and upto 3-fold increased in the ischemic hemisphere. The presence ofneutrophils was positively correlated with the degranulation state ofthe cells consistent with mast cell morphology in the same brains. It istherefore conceived that mast cell-derived chemokines participate inbuilding the chemotactic gradient up which neutrophils are migratinginto the tissue. This can also increase the likelihood of floriderythrocyte extravasation leading to hemorrhagic tissue changes.

By using substances, such as cromoglycate, whose pharmacological effectconsists essentially exclusively of affecting the degranulation oractivation of mast cells or inhibiting the main granula constituents ofcerebral mast cells, it is possible to protect and treat complications,such as brain edema and hemorrhagic brain insults, caused by thebreaking down of the brain-blood barrier during thrombolysis. For thepurpose of the present invention, cromoglycate and the similarcompounds, such as any 2-carboxylatomchromon-5′-yl-2-hydroxypropanederivatives, nedocromil and tranilast, are considered to be agents,which specifically prevent or reduce mast cell degranulation oractivation, in the sense that they do not have other major tissueactivity except for that exhibited through their mast cell degranulationor activation blocking mechanism. Similarly, as will be discussed inmore detail below, inhibitors of the c-kit receptors on mast cells aswell as chymase and procollagenase activators can be used in the presentinvention.

The present invention provides new thrombolytic compositions, whichcontain a therapeutically effective amount of a mast celldegranulation-blocking and/or mast cell activation-blocking agent, toprevent cerebral complications, such as brain edema or hemorrhagic braininsults, in a patient during or after thrombolysis.

The invention also comprises the use of a mast celldegranulation-blocking and/or mast cell activation-blocking agent in themanufacture of a medicament for preventing or reducing mast celldegranulation in a patient subjected to thrombolysis.

In the following, the term “mast cell degranulation-blocking agent” willbe used interchangeably with “mast cell degranulation-blocking and/ormast cell activation-blocking agent” to designate an agent having eitheror both of the two activities: mast cell degranulation blocking and mastcell activation blocking. Preferably the activity is, as discussedabove, specific. Within the above definition are also included compoundsacting primarily on c-kit receptor responsible of mast cell maturationand activation, and its intracellular signaling pathway.

The invention further provides efficient precaution against cerebraledematous change in thrombolytic conditions conducive to cerebralischemia. The treatments according to the invention involve theadministration of a therapeutically effective amount of a mast celldegranulation-blocking and/or mast cell activation-blocking agent

By the present invention, significant reductions in serious hemorrhagic,especially cerebral complications, in thrombolytic treatments, can bereached. The reduction of ischemic edema is up to 40% compared tocontrol can be obtained (this figure is based on the animal experimentsdescribed in detail below). The finding was surprising, since it haslong been held that the immediate phase of brain edema is caused bycytotoxic mechanism leading to a shift of extracellular fluid, notintravascular fluid, into the cerebral cellular compartment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar chart showing the percentual hemispheric expansioncaused by focal cerebral ischemia in rats after pretreatment with twodifferent substances in comparison to control;

FIG. 2 is a bar chart showing the fluorescence above autofluorescencethreshold for the same samples as in FIG. 1.

FIGS. 3 to 6 are further bar charts showing the results of Examples 2and 3.

DETAILED DESCRIPTION OF THE INVENTION

Cerebral mast cells degranulate to some extent during ischemic strokeand their degranulation is further increased by r-TPA treatment. Mastcell granules contain heparin, and are the only cell type of the bodycapable of heparin synthesis. Should there occur subtle breaches ofblood-brain barrier to allow red blood cell extravasation, the heparinreleased by mast cells may prevent the formation of fibrin fibres forthe platelets to attach and form a plug preventing furtherextravasation. Furthermore, mast cells release chymase, which is apotent proteolytic substance and lyses the fibronectin of the vascularbasal membrane.

Plasmin can also activate neutrophil-derived metalloproteinases thatlyse collagen. These mechanisms may give rise to lysis of two of thethree major components (laminin, collagen, fibronectin) of the vascularbasement membrane. These events can initiate local bleedings and evensolid parenchymal hematomas, if this phenomenon occurs in the vicinityof small arterial vessels such as the penetrating arteries in the basalganglia and thalamus. These are well-known predilection areas of thepresence of cerebral mast cells as well as spontaneous cerebralbleedings.

Although many of the triggering factors for mast cell degranulation havebeen documented, the major trigger in cerebral ischemia remains unknown.One mechanism may relate to complement protein activation, formation ofanaphylatoxins C3a and C5a, which are among the most potent activatorsof mast cells. The present inventors have previously demonstrated thatterminal ischemic and hemorrhagic brain insults produce activation ofthe terminal complement pathway, which involves the synthesis ofanaphylatoxins. Interestingly, it was demonstrated that even in vitromixing of ex vivo human plasma and cerebrospinal fluid led to complementactivation. Therefore, even temporary and subtle initial breaches of theblood-brain barrier early during ischemia might produce C3a and C5a andtrigger mast cell degranulation and more florid BBB damage and eventualbrain edema. Other mechanisms could include production of cytokines suchas interleukins of tumor necrosis factor-α.

Since r-TPA increased the mast cell degranulation both in vitro and invivo, inhibitors of mast cell degranulation and activation might proveeffective in preventing a fraction of the feared hemorrhagiccomplications of thrombolytic treatment, especially in the brain.Furthermore, mast cell stabilizing therapies might attenuate thereperfusion injury caused by neutrophils infiltrating the brainparenchyma. This is a common sequel in ischemic stroke even in theabsence of thrombolysis. Furthermore, application of these therapiesmight help to increase the fraction of patients that today can beconsidered eligible for thrombolytic therapy.

Practical applications of the present invention include:

I. Prevention of Serious Hemorrhagic, Especially Cerebral Complications,in the Thrombolytic Treatment

Typical applications include

1) acute ischemic stroke

2) acute myocardial infarction

3) massive pulmonary embolism

4) occlusion of peripheral arteries

5) thrombolysis of intracerebroventricular haematoma

II. Prevention of Reperfusion Injury, Inflammatory Changes and BrainEdema Associated with Recanalization of Cerebral Artery OccurringSpontaneously or after Thrombolysis

According to the invention, a mast cell degranulation-blocking or mastcell activation-blocking agent is employed. The agent may have either orboth of these activities. The aim is in particular to stabilize the mastcells by, e.g. preventing degranulation of the cells and the release ofsubstances contain in the granulas. Generally speaking, activation ofmast cells is to be avoided within the scope of the present invention.

The mast cell degranulation-blocking or mast cell activation-blockingagent can be administered separately from the thrombolytically activecomponent. Preferably, it is however incorporated into the thrombolyticcomposition.

If administered separately, the mast cell degranulation-blocking or mastcell activation-blocking agent should preferably be administered before,preferably at least 5 minutes, in particular at least 10 minutes beforethe patient is subjected to thrombolysis.

If incorporated into the thrombolytical composition, the agent ispresent in a therapeutically effective amount to prevent or reduce anycerebral complications, such as brain edema, in a patient subjected tothrombolytic treatment.

The mast cell degranulation-blocking (including mast cellactivation-blocking) agent is, according to the invention, preferablyselected from the group of2-carboxylatochromon-5′-yl-2-hydroxypropanederivatives and histamine-1receptor antagonists. Examples of mast cell degranulation-blockingagents of the first group are bis(acetoxymethyl) cromoglycate, disodiumcromoglycate and nedocromil. Further compounds exhibiting selective mastcell degranulation/activation blocking effect include tranilast, andcompounds acting primarily on (inhibiting) the c-kit receptorresponsible of mast cell maturation and activation, such as imatinibe(e.g. in the form of its mesylate salt).

Examples of the histamine-1 inhibitors are: azatadine, azelastine,burfroline, cetirizine, cyproheptadine, doxantrozole, etodroxizine,forskolin, hydroxyzine, ketotifen, oxatomide, pizotifen, proxicroril,N,N′-substituted piperazines and terfenadine.

Further examples include flavonoids, which inhibit mast cell secretionand proliferation. These are exemplified by quercetin optionally incombination with the proteoglycan chondroitin sulphate. Histamine-2receptor antagonists, such as cimetidine, optionally combined with e.g.hydroxyzine, along with indolinone derivatives, and IPD-1151T are otherexamples.

The present invention also provides for prevention of mast cell-inducedblood-brain barrier damage by inhibition of the main granuleconstituents of cerebral mast cells, which lyse basal lamina proteins,such as chymase, and procollagenase activators. Thus, substancesspecifically inhibiting chymase and procollagenase activators, such asTIMP (tissue inhibitors of metalloproteinase 1) are included, inparticular substances capable of penetrating the blood-brain barrier.Even compounds capable selectively of inhibiting the histamine releasedfrom mast cells should be considered.

The mast cell degranulation-blocking and/or mast cellactivation-blocking agent is administered in a therapeutically efficientamount. Typically, that amount is about 0.05 to 100 milligrams perkilogram body weight of the patient.

The present invention provides for new thrombolytic compositions, whichcontain a component preventing or alleviating cerebral complicationscaused by the active components. The thrombolytically active componentis typically selected from the group of alteplase, tenecteplase,reteplase, and streptochinase. As examples of commercial compositionswhich can be complemented with the agent, the following can bementioned: Actilyse (supplier Boehringer Ingelheim), Metalyse (supplierBoehringer Ingelheim), Rapilysin (supplier: Roche) and Streptase(supplier: Aventis Behring). Such a compositions are, according to theinvention, complemented with a suitable amount of a mast celldegranulation-blocking and/or mast cell activation-blocking agent. Theamount is generally 0.01 to 100 mg/l. The thrombolytic compositions areformulated for parenteral administration.

When separately administered, the compounds employed in the methods ofthe present invention may be administered by any means that results inthe contact of the active agent with the agent's site of action in thebody of a patient. The compounds may be administered by any conventionalmeans available for use in conjunction with pharmaceuticals, either asindividual therapeutic agents or in a combination of therapeutic agents.For example, they may be administered as the sole active agent in apharmaceutical composition, or they can be used in combination withother therapeutically active ingredients.

The compounds may be combined with a pharmaceutical carrier selected onthe basis of the chosen route of administration and standardpharmaceutical practice as described, for example, in Remington'sPharmaceutical Sciences (Mack Pub. Co., Easton, Pa., 1980), thedisclosures of which are hereby incorporated herein by reference, intheir entireties.

Compounds of the present invention can be administered to a mammalianhost in a variety of forms adapted to the chosen route ofadministration, e.g., orally or parenterally. Parenteral administrationin this respect includes administration by the following routes:intravenous, intramuscular, subcutaneous, rectal, intraocular,intrasynovial, transepithelial including transdermal, ophthalmic,sublingual and buccal; topically including ophthalmic, dermal, ocular,rectal, and nasal inhalation via insufflation aerosol. The activecompound may be orally administered, for example, with an inert diluentor with an assimilable edible carrier, or it may be enclosed in hard orsoft shell gelatin capsules, or it may be compressed into tablets, or itmay be incorporated directly with the food of the diet. For oraltherapeutic administration, the active compound may be incorporated withexcipient and used in the form of ingestible tablets, buccal tablets,troches, capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations should preferably contain at leastabout 0.1% of active compound. The percentage of the compositions andpreparations may, of course, be varied and may conveniently be, forexample, from about 2 to about 6% of the weight of the unit. The amountof active compound in such therapeutically useful compositions ispreferably such that a suitable dosage will be obtained. Preferredcompositions or preparations according to the present invention may beprepared so that an oral dosage unit form contains from about 0.1 toabout 1000 mg of active compound, and all combinations andsubcombinations of ranges and specific amounts therein.

The tablets, troches, pills, capsules and the like may also contain oneor more of the following: a binder, such as gum tragacanth, acacia, cornstarch or gelatin; an excipient, such as dicalcium phosphate; adisintegrating agent, such as corn starch, potato starch, alginic acidand the like; a lubricant, such as magnesium stearate; a sweeteningagent such as sucrose, lactose or saccharin; or a flavoring agent, suchas peppermint, oil of wintergreen or cherry flavoring. When the dosageunit form is a capsule, it may contain, in addition to materials of theabove type, a liquid carrier. Various other materials may be present ascoatings or to otherwise modify the physical form of the dosage unit.For instance, tablets, pills, or capsules may be coated with shellac,sugar or both. A syrup or elixir may contain the active compound,sucrose as a sweetening agent, methyl and propylparabens aspreservatives, a dye and flavoring, such as cherry or orange flavor. Ofcourse, any material used in preparing any dosage unit form ispreferably pharmaceutically pure and substantially non-toxic in theamounts employed. In addition, the active compound may be incorporatedinto sustained-release preparations and formulations.

The active compound may also be administered parenterally orintraperitoneally. Solutions of the active compound as a free base or apharmacologically acceptable salt can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. A dispersioncan also be prepared in glycerol, liquid polyethylene glycols andmixtures thereof and in oils. Under ordinary conditions of storage anduse, these preparations may contain a preservative to prevent the growthof microorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form is preferably sterile and fluid toprovide easy syringability. It is preferably stable under the conditionsof manufacture and storage and is preferably preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier may be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, liquidpolyethylene glycol and the like), suitable mixtures thereof, andvegetable oils. The proper fluidity can be maintained, for example, bythe use of a coating, such as lecithin, by the maintenance of therequired particle size in the case of a dispersion, and by the use ofsurfactants. The prevention of the action of microorganisms may beachieved by various antibacterial and antifungal agents, for example,parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.In many cases, it will be preferable to include isotonic agents, forexample, sugars or sodium chloride. Prolonged absorption of theinjectable compositions may be achieved by the use of agents delayingabsorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompound in the required amount, in the appropriate solvent, withvarious of the other ingredients enumerated above, as required, followedby filtered sterilization. Generally, dispersions may be prepared byincorporating the sterilized active ingredient into a sterile vehiclethat contains the basic dispersion medium and the required otheringredients from those enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation may include vacuum drying and the freeze dryingtechnique, which yield a powder of the active ingredient, plus anyadditional desired ingredient from the previously sterile-filteredsolution thereof.

The therapeutic compounds of this invention may be administered to apatient alone or in combination with a pharmaceutically acceptablecarrier. As noted above, the relative proportions of active ingredientand carrier may be determined, for example, by the solubility andchemical nature of the compound, chosen route of administration andstandard pharmaceutical practice.

The dosage of the compounds of the present invention that will be mostsuitable will vary with the form of administration, the particularcompound chosen and the physiological characteristics of the particularpatient under treatment.

In particular, adminstration can be carried out parenterally, forexample by i.v., i.c.v. (intracerebroventricularly) and i.m.administration. Parenteral compositions usually contain a bufferingagent and, optionally, a stabilizing agent.

When necessary, in order to promote penetration of theblood-brain-barrier, the active compounds can be administered by usingvarious known strategies for gaining drug access to the brain. Theseinclude the transcellular lipophilic pathway, which allows small,lipophilic compounds to cross the blood-brain barrier. A second pathwayis “receptor-mediated endocytosis. Further, as known in the art, someexperimental work has shown that a monoclonal antibody for thetransferrin receptor, coupled with brain-derived neurotrophin factor,which is neuroprotective but cannot cross the barrier itself, can bothcross the barrier and exert neuroprotective effects. Endothelial cellsof the blood-brain barrier also express a number of transport proteins,including transporters for glucose, amino acids, nucleosides, and othercompounds. Thus, to focus on the latter strategy, the compounds can bedesigned such that they gain access to the brain by going through thesetransport processes. It is, however, also possible to block theseprocesses, in that way bolstering brain levels of endogenous permeant.

For the sake of completeness, it should be pointed out that thecompounds employed in the uses and methods of the present invention mayexist in prodrug form. As used herein, the term “prodrug” is intended toinclude any covalently bonded carriers which release the active parentdrug or other formulas or compounds employed in the methods of thepresent invention in vivo when such prodrug is administered to amammalian subject. Since prodrugs are known to enhance numerousdesirable qualities of pharmaceuticals (e.g., solubility,bioavailability, manufacturing, etc.), the compounds employed in thepresent methods may, if desired, be delivered in prodrug form. Thus, thepresent invention contemplates methods of delivering prodrugs. Prodrugsof the compounds employed in the present invention may be prepared bymodifying functional groups present in the compound in such a way thatthe modifications are cleaved, either in routine manipulation or invivo, to the parent compound.

Accordingly, prodrugs include, for example, compounds described hereinin which a hydroxy, thiol, amino, or carboxy group is bonded to anygroup that, when the prodrug is administered to a mammalian subject,cleaves to form a free hydroxyl, thiol, free amino, or carboxylic acid,respectively. Examples include, but are not limited to, acetoxyalkyls,acetate, formate and benzoate derivatives of alcohol, thiol, and aminefunctional groups; and alkyl, carbocyclic, aryl, and alkylaryl esterssuch as methyl, ethyl, propyl, iso-propyl, butyl, isobutyl, sec-butyl,tert-butyl, cyclopropyl, phenyl, benzyl, and phenethyl esters, and thelike.

To make drug delivery route more practical especially in the clinicalsetting, rat MCAO model can be used as described in detail in thisapplication by replacing i.c.v. drug administration as the pretreatmentbefore thrombolysis with intravenous (i.v.) drug administration of analternative mast cell degranulation-blocking and/or mast cellactivation-blocking agent, which possess the capacity to penetratethrough blood-brain barrier more effectively than sodium cromoglycate.

Next, the invention will be illustrated with examples:

EXAMPLE 1 Methods

The suture filament model was used to induce focal cerebral ischemia for60 min in rats. Reperfusion was allowed for 3 h, at which point the ratswere killed, cardioperfused and their brains were dissected into coronalsections. Evans Blue-albumin (2%, 0.3 ml/100 g) a fluorescent dye, wasinjected i.v. 20 min before termination to monitor BBB (blood brainbarrier) permeability. TTC (2,3,5-triphenyltetrazolium chloride, 2%)staining was used to quantitate the infarct volumes. The volumetricexpansion of the ischemic hemisphere was quantitated with computerizedplanimetry. Rats were assigned in three pharmacological treatments: mastcell stabilizer disodium cromoglycate 750 ug in 10 ul i.c.v. (does noteasily cross BBB) (n=14) or control (10 ul saline i.c.v.) (n=13) 5 minprior to ischemia, and a mast cell degranulation agent, compound 48/80(n=11) administered (0.025 mg i.v.) 3 min prior to reperfusion. TheEvans Blue extravasation was analysed using fluorescence microscopy andcomputerized image analysis-based quantitation of the fluorescent pixelsin five random regions of interest.

Results:

The volumetric hemispheric expansion caused by ischemia was highlysignificantly influenced by pharmacologic modulation of mast celldegranulation (Kruskal-Wallis ANOVA p<0.001, FIG. 1), as well as themean number of fluorescent pixels indicating rates of extravasation(p<0.001, FIG. 2). Corrected infarct volumes were not influenced by thetreatments: control 189±31, cromoglycate 252±41, compound 48/80 193±24mm³, p=0.33.

The results confirm the observations discussed above: inhibition of mastcell degranulation by intraventricular administration of cromoglycateled to a 39% reduction of the acute-phase ischemic edema.

EXAMPLE 2

Rats were given 10 mg/kg of r-TPA, which is considered equivalent to thetypical dose given to humans in thrombolysis in stroke and myocardialinfarction. Two timings of r-TPA were used to investigate in rat brainthe hemorrhagic conversion of infarction, the single most seriouscomplication and hazard in clinical thrombolytic therapy. The grouptermed r-TPA+saline received 1-hour infusiuon of r-TPA starting 5minutes (10% as bolus) prior to reperfusion after 90 min of middlecerebral artery occlusion (MCAO), whereas the group saline+r-TPAreceived r-TPA starting 1 hour after the reperfusion after 90 min ofMCAO. Both timings resulted in significant hemorrhage formation, ascompared to sham operated or saline-treated controls. Brains weredissected at 4.5 hours after reperfusion. Areas of hemorrhage weremeasured by microscopical image analysis on six histological sectionscut through the hemispheres and added together (cf. FIG. 3)

The polymorphonuclear leukocytes (neutrophils, shown in bars, leftY-axis, of FIG. 4) were calculated on the section encompassing the basalganglia, infarct core and penumbra. While both groups that receivedr-TPA showed significantly increased neutrophil density compared tosham-operated rats, those which received only saline did not. r-TPAincreased neutrophil counts even in the contralateral non-infarctedhemisphere. In the ipsilateral hemisphere, neutrophils appeared to belargely emigrated especially in the saline-treated rats (scatter plot,right Y-axis).

Rats were treated either with saline or cromoglycate i.c.v. andsubjected to 90 min of MCAO. They were also treated with 10 mg/kg ofr-TPA in a 1-hour infusion starting 2 min (10% as bolus) beforereperfusion, a dose equivalent to that used clinically in thrombolysisof human stroke and myocardial infarction. Brains were dissected at 4.5hours after reperfusion. The amount of brain edema was measured byquantitating the areas of hemispheres on histological sections withmicroscopical image analysis. The extent of hemispheric expansion (areascorrected by the size of infarctions) indicative of brain edema wasfound to be 54% reduced by cromoglycate (FIG. 5).

In the lower panel, the total area of hemorrhagic conversion wasquantitated in six sections cut through the hemispheres. The i.c.v.cromoglycate treatment was found to decrease the area of hemorrhagesignificantly by 74%.

EXAMPLE 3

Additional data was derived from experiments using mast cell-deficientWsRc^(Ws/Ws) rats carrying a defective gene for c-kit (ligand for stemcell factor [SCF]) required for mast cell differentiation) and theirwild-type littermates. Animals were subjected to transient focalcerebral ischemia and treated with r-TPA as described above. In the mastcell deficient rats, the mean area of hemorrhage measured on the midlinehistological brain section was 142625:m² and 7100:m² in the wild-typelittermates (p<0.01).

FIG. 6 shows the results obtained for MC-deficient gene manipulated ratsand their non-manipulated littermates subjected to 90 min of MCAO and4.5 hours of reperfusion. The density of neutrophils were counted in thetissue section encompassing basal ganglia, the infarct core andpenumbra. The number of neutrophils was significantly reduced in theMC-deficient rats, especially in the infarct core and basal ganglia. Inthe lower panel it is indicated that the reduction in the pool ofextravasated neutrophils was statistically most consistent. We performadditional analyses that will indicate the effect of cromoglycatetherapy on the r-TPA-related neutrophil response, which is a componentof reperfusion injury after recanalization of an occluded cerebralartery observed in FIG. 4.

The results presented indicate that interventions preventing mast celldegranulation (sodium cromoglycate as the model compound), as well asthose influencing the function of c-kit receptor required for mast cellmaturation, markedly reduce the hemorrhagic conversion when treatingischemic stroke with thrombolytic therapy. Furthermore, the resultingbrain expansion is reduced by cromoglycate. Also the aggravation ofinflammatory response, the neutrophil accumulation, is significantlyreduced by sodium cromoglycate and c-kit knock-out. Therefore, mast cellinhibiting adjunct treatments would probably significantly improve thesafety of thrombolytic therapies in man.

EXAMPLE 4

Thrombolysis with r-TPA of spontaneous intraventricular bleedings is anexperimental treatment in humans, which accelerates the removal ofdetrimental blood clots from cerebral ventricles and subarachnoidal CSFspaces. However, the handicap of this treatment is that it more thandoubles the rates of secondary brain hemorrhages in preliminary humanexperiments. This can cause fatal outcome and additional postoperativeneurological disability. To eliminate this handicap, experiments can beperformed in rats, which are subjected to intracerebroventricularhemorrhages induced by stereotactical infusion of autologous arterialblood. Comparisons are made of the rates of secondary cerebralhemorrhages in groups treated with a prolonged intraventricularr-TPA-infusion with and without prior and concomitant i.c.v.administration of sodium cromoglycate. The volume of hemorrhage and therate of new intracerebral hemorrhages secondary to the r-TPA can bemonitored by serial MRI (T2*sequence) and the total volume ofhemorrhagic lesions in serial histological sections determined.

1. A method for decreasing intracerebral hemorrhaging in a cerebralischemia patient subjected to tissue plasminogen activator (TPA)treatment, comprising administering to said patient a therapeuticallyeffective amount of a composition comprising a mast cell activation- ordegranulation-blocking agent selected from the group consisting ofbis(acetoxymethyl) cromoglycate, disodium cromoglycate, nedocromil andtranilast.
 2. The method according to claim 1, wherein the mast cellactivation- or degranulation-blocking agent is administered in an amountof about 0.05 to 100 milligrams per kilogram body weight of the patient.3. The method according to claim 1, wherein said composition isformulated for parenteral administration.
 4. The method according toclaim 1, wherein said composition further comprises a thrombolyticagent.
 5. The method according to claim 1, wherein the mast cellactivation- or degranulation-blocking agent is disodium cromoglycate.